Recently developed techniques of molecular cloning make it possible to clone a nucleotide sequence which encodes a protein and to produce that protein in quantity using a suitable host-vector system (Maniatis, T., et al., Molecular Cloning--A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982). The protein can then be recovered by known separation and purification techniques. Cloning methods which have been used to date can be grouped into three general categories: (1) methods based upon knowledge of the protein structure, for example, its amino acid sequence; (2) methods based upon identification of the protein expressed by the cloned gene using an antibody specific for that protein; and (3) methods based upon identification of an RNA species which can be translated to yield the protein or activity encoded by the gene of interest.
Each of these classes of methods becomes difficult to apply when the protein of interest (and its corresponding mRNA) is produced in very low amount. Thus, if it is difficult to obtain an adequate quantity of purified protein, then it is difficult to determine the amino acid sequence of the protein. Similarly, identification of an expressed protein by antibody binding is preferentially carried out using a high-titer monospecific polyclonal antiserum. Such an antiserum cannot be obtained in the absence of quantities of the pure protein (antigen). A monoclonal antibody offers an alternative approach, but the required antibody can also be difficult to obtain in the absence of suitable antigen, and such monoclonal antibody may not react with the protein in the form in which the protein is expressed by available recombinant host-vector systems. Finally, translation of an RNA species to yield an identifiable protein or activity requires that the RNA in question be present in the RNA source in sufficient abundance to give a reliable protein or activity.
Among the methods listed above, RNA translation has been the most generally applicable procedure for identifying cDNA clones corresponding to rare mRNAs. In a common embodiment of this procedure, cDNA clones carrying sequences complementary to specific mRNAs are identified by hybridization selection. The cloned DNAs are denatured individually or in groups, immobilized on a solid matrix, and hybridized to preparations of mRNA. The RNA-DNA duplex is heated to release the mRNA, which is then translated in cell-free, protein-synthesizing systems or in Xenopus oocytes. The translation products are, identified by immunoprecipitation and/or SDS-polyacrylamide gel electrophoresis or by biological assays.
A serious limitation to the use of RNA translation methods is the difficulty of obtaining an adequate signal from the translation product of a rare mRNA. It may be possible to enrich for a desired mRNA by various procedures; two principal methods are size fractionation and removal of RNA sequences which are shared with RNA preparations from cells which do not produce the protein of interest. However, detection of a translation product ultimately depends upon the sensitivity of the assay for the protein of interest. In the case where an RNA is rare and the assay for its translation product is relatively insensitive, the amount of protein produced by oocyte translation may be below the threshold of detection in the assay.
A more direct approach to isolation of specific cDNA clones could be based upon identification of protein products of cDNA clones in an expression vector. An expression vector is a self-replicating DNA element which contains signals for efficient transcription and translation of a cDNA which is inserted into it. In the past, expression vectors have mainly been used for manufacturing quantities of protein after isolation of a clone by traditional means. As noted above, RNA translation methods produce proteins according to the abundance of specific mRNAs, so that a rare mRNA may generate an undetectable quantity of protein. Expression vectors, by contrast, express the protein product of each cDNA insert at an equally high level. Thus the product of a rare mRNA will in general be more easily detected using an expression vector rather than RNA translation.
Cloning by expression has been carried out previously in a general sense in bacteria and in yeast. For example, complementation of a bacterial or a yeast mutation by transformation with an appropriate clone library has been used to isolate a number of microbial genes. Attempts at antibody identification of mammalian protein expressed in a bacterial host-vector system, as described above, constitute another example of this approach. However, mammalian proteins which are of commercial interest, particularly as therapeutic agents, are often secreted proteins containing carbohydrates and/or numerous disulphide bonds. Because of these attributes, it is often the case that a mammalian protein synthesized in a microbial host-vector system is expressed in an inactive form and/or with altered antigenic determinants and thus cannot be identified by activity or antibody assays.
The use of a mammalian host-vector system for expression cloning of mammalian cDNAs has been attempted in the past (Okayama, H. and Berg, P. 1983 Mol. Cell Biol. 3 280-289). Mammalian host-vector systems employing transient expression of genes in COS-1 cells have often been used to verify the identity of cDNA clones isolated by standard techniques (e.g. Gray et al 1982 Nature 295 503-508; Yokota et al 1984 Proc. Nat. Acad. Sci. 81 1070-1074). However, there is no report in the literature of a cDNA clone identified directly by the activity of its protein produced in a mammalian expression system.
Thus, it can be seen that it would be desirable to have a method for isolation of specific cDNA clones by assay of proteins produced in a mammalian expression system, particularly for cloning cDNAs corresponding to rare mRNAs.